Protein synthesis is a fundamental biological process that underlies the development of polypeptide therapeutics, diagnostics, and industrial enzymes. With the advent of recombinant DNA (rDNA) technology, it has become possible to harness the catalytic machinery of the cell to produce a desired protein. This can be achieved within the cellular environment or in vitro using extracts derived from cells.
Over the past decade, the productivity of cell-free systems has improved 2-orders of magnitude, from about 5 μg/ml-hr to about 500 μg/ml-hr. This accomplishment has made in vitro protein synthesis a practical technique for laboratory-scale research and provides a platform technology for high-throughput protein expression. It also begins to suggest the feasibility for using cell-free technologies as an alternative means to in vivo large-scale production of protein pharmaceuticals.
Cell-free protein synthesis offers several advantages over conventional, in vivo, protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall and membrane components in vitro is advantageous since it allows for control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. The redox potential, pH, or ionic strength can also be altered with greater flexibility than in vivo since we are not concerned about cell growth or viability. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved.
In vitro translation is also recognized for its ability to incorporate unnatural and isotope-labeled amino acids as well as its capability to produce proteins that are unstable, insoluble, or cytotoxic in vivo. In addition, cell-free protein synthesis may play a role in revolutionizing protein engineering and proteomic screening technologies. The cell-free method bypasses the laborious processes required for cloning and transforming cells for the expression of new gene products in vivo, and is becoming a platform technology for this field.
Despite all of the promising features of cell-free protein synthesis, its practical use and large-scale implementation has been limited by several obstacles. Paramount among these are short reaction times and low protein production rates, which lead to poor yields of protein synthesis and excessive reagent cost. The pioneering work of Spirin et al. (1988) Science 242:1162-1164 initially circumvented the short reaction times problem with the development of a continuous flow system. Many laboratories have duplicated and improved upon this work, but they have all primarily used methods that constantly supply substrates to the reaction chamber. This approach increases the duration of the translation reaction and protein yield as compared to the batch system. However, it is inefficient in its use of expensive reagents, generally produces a dilute product, and has not provided significant improvements in production rates.
The conventional batch system offers several advantages over these continuous and semi-continuous schemes, which include ease of scale-up, reproducibility, increased protein production rates, convenience, applicability for multi-plexed formats for high throughput expression, and more efficient substrate use. These advantages make improving the batch system productivity crucial for the industrial utilization of cell-free protein synthesis. Recently, a series of findings have been reported which begin to elucidate the causes of early termination of protein synthesis in batch reactions. Furthermore, Kim and Swartz (2001) Biotechnol Bioeng. 74:309-316; Kim and Swartz (1999) Biotechnol Bioeng. 66:180-188 have illustrated that the length of the conventional batch reaction could be extended from 20 minutes to up to 2 hours with the use of novel energy regeneration systems. While these approaches are promising, there is still a tremendous need for developing an economically viable commercial process. Increasing the product yield by improving the protein production rate and extending the reaction time is an essential component of filling this need. Reducing the cost of protein synthesis reagent, especially the chemical energy source, is another important component.